Stabilization of alpha-amylases towards calcium depletion and acidic pH

ABSTRACT

The present invention relates to variants of a parent alpha-amylase, the variant having improved stability or activity at low calcium conditions or at low pH.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/005,667filed Jan. 25, 2016, now U.S. Pat. No. 9,394,532, which is a division ofU.S. application Ser. No. 14/525,877 filed Oct. 28, 2014, now U.S. Pat.No. 9,273,297, which is a division of U.S. application Ser. No.13/518,962 filed Jun. 25, 2012, now U.S. Pat. No. 8,900,848, which is a35 U.S.C. 371 national application of PCT/EP2011/050074 filed Jan. 4,2011 which claims priority or the benefit under 35 U.S.C. 119 ofEuropean application nos. 10150063.5 and 10150062.7 filed Jan. 4, 2010and Jan. 4, 2010 and U.S. provisional application Nos. 61/292,324,61/292,327, 61/304,092, 61/333,930, 61/354,775, 61/354,817, 61/355,230and 61/362,536 filed Jan. 5, 2010, Jan. 5, 2010, Feb. 12, 2010, May 12,2010, Jun. 15, 2010, Jun. 15, 2010 Jun. 16, 2010 and Jul. 8, 2010 thecontents of which are fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to variants of an alpha-amylase havingimproved stability at an acidic pH and/or in the presence of strongchelators compared to its parent enzyme. Further, the invention relatesto nucleic acids encoding the variants, methods of producing thevariants, and methods for using the variants.

BACKGROUND OF THE INVENTION

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyzes hydrolysis of starch andother linear and branched 1,4-gluosidic oligo- and polysaccharides.

There is a long history of industrial use of alpha-amylases in severalknown applications such as detergent, baking, brewing, starchliquefaction and saccharification, e.g., in preparation of high fructosesyrups or as part of ethanol production from starch. These and otherapplications of alpha-amylases are known and utilize alpha-amylasesderived from microorganisms, in particular bacterial alpha-amylases.

Among the first bacterial alpha-amylases to be used was an alpha-amylasefrom B. licheniformis, also known as Termamyl, which has beenextensively characterized and the crystal structure has been determinedfor this enzyme. Alkaline amylases, such as AA560 (SEQ ID NO: 2),disclosed in WO 00/60060, form a particular group of alpha-amylases thathave found use in detergents. Many of these known bacterial amylaseshave been modified in order to improve their functionality in aparticular application.

Termamyl and many highly efficient alpha-amylases required calcium foractivity. In the crystal structure for Termamyl it was found that fourcalcium atom were bound in the alpha-amylase structure coordinated bynegatively charged amino acid residues. This requirement for calcium isa disadvantage in applications where strong chelating compounds arepresent, such as in detergents or during ethanol production from wholegrains, where plant material comprising high amount of natural chelaterssuch as phytate is hydrolysed using alpha-amylases.

Calcium-insensitive amylases are known, e.g., the alpha-amylasesdisclosed in EP 1022334 and WO 03/083054, and a Bacillus circulansalpha-amylase having the sequence disclosed in UNIPROT:Q03657, but theseamylases are inferior to many of the calcium-sensitive amylase when itcomes to starch hydrolysis and starch removal in various applications.

It would therefore be beneficial to provide variants of acalcium-sensitive alpha-amylase with reduced calcium sensitivitycompared to its parent enzyme.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of a parentTermamyl-like alpha-amylase, comprising an alteration at two, three,four or five positions corresponding to positions 163, 188, 205, 208 and209 of amino acids 1 to 485 of SEQ ID NO: 2 wherein the alteration(s)are independently

(i) an insertion of an amino acid immediately downstream of theposition,

(ii) a deletion of the amino acid which occupies the position, and/or

(iii) a substitution of the amino acid which occupies the position, and

wherein the variants have alpha-amylase activity.

The variants of the invention may further comprise one or moreadditional substitution(s).

Additionally, the isolated variants may comprise further alterationsknown to improve the performance of alpha-amylases including a deletioncorresponding to amino acids 183 and 184 and substitutions in one ormore of the positions 186, 193, 195, 202, 206, 214, 244, 452, 474 and475, and each position corresponds to a position of the amino acidsequence of the enzyme having the amino acid sequence of SEQ ID NO: 2.

The variants of the invention have reduced calcium sensitivity comparedwith the parent alpha-amylase.

The present invention also relates to isolated nucleotide sequencesencoding the variant alpha-amylases or polypeptides having alpha-amylaseactivity and to nucleic acid constructs, vectors, and host cellscomprising the nucleotide sequences.

Methods for preparing the variants of the invention are also provided.

The present invention also relates to compositions comprising thevariants of the invention, in particular a detergent additivecomposition, detergent composition, composition for manual or automaticdishwashing or compositions for manual or automatic laundry washing.Further, the invention relates to the use of an alpha-amylase variantaccording to the invention for washing and/or dishwashing, textiledesizing and starch liquefaction. The invention also relates to a methodfor producing ethanol or other chemicals using the variant of theinvention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its polypeptideproduct. The boundaries of the coding sequence are generally determinedby an open reading frame, which usually begins with the ATG start codonor alternative start codons such as GTG and TTG and ends with a stopcodon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a variant ofthe present invention. Each control sequence may be native or foreign tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of the variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to additional nucleotides that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide; wherein the fragment has alpha-amylase activity.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved pH stability: The term “improved pH stability” is definedherein as a variant enzyme displaying retention of enzymatic activityafter a period of incubation at a particular pH, which reduces theenzymatic activity of the parent enzyme. Improved pH stability may alsoresult in variants better able to catalyze a reaction under such pHconditions.

Isolated variant: The terms “isolated” and “purified” mean a polypeptideor polynucleotide that is removed from at least one component with whichit is naturally associated. For example, a variant may be at least 1%pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, atleast 40% pure, at least 60% pure, at least 80% pure, and at least 90%pure, as determined by SDS-PAGE and a polynucleotide may be at least 1%pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, atleast 40% pure, at least 60% pure, at least 80% pure, at least 90% pure,and at least 95% pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Parent Enzyme: The term “parent” alpha-amylase as used herein means analpha-amylase to which modifications, e.g., substitution(s),insertion(s), deletion(s), and/or truncation(s), are made to produce theenzyme variants of the present invention. This term also refers to thepolypeptide with which a variant is compared and aligned. The parent maybe a naturally occurring (wild type) polypeptide, or it may be a variantthereof, prepared by any suitable means. For instance, the parentprotein may be a variant of a naturally occurring polypeptide which hasbeen modified or altered in the amino acid sequence. A parent may alsobe an allelic variant which is a polypeptide encoded by any of two ormore alternative forms of a gene occupying the same chromosomal locus.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′- and/or 3′-end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having alpha-amylase activity.

Variant: The term “variant” is defined herein as an alpha-amylasecomprising one or more alterations, such as substitutions, insertions,deletions, and/or truncations of one or more specific amino acidresidues at one or more specific positions in the polypeptide.

Wild-Type Enzyme: The term “wild-type” alpha-amylase denotes analpha-amylase expressed by a naturally occurring microorganism, such asan yeast or filamentous fungus found in nature.

Conventions for Designation of Variants

In the present invention, a specific numbering of amino acid residuepositions in the alpha-amylase variants is employed. For example, byaligning the amino acid sequences of known alpha-amylases, it ispossible to designate an amino acid position number to any amino acidresidue in any alpha-amylase enzyme.

Using the numbering system originating from the amino acid sequence ofthe alpha-amylase disclosed in SEQ ID NO: 2, aligned with the amino acidsequence of a number of other alpha-amylases, it is possible to indicatethe position of an amino acid residue in an alpha-amylase in regions ofstructural homology.

In describing the various alpha-amylase variants of the presentinvention, the nomenclature described below is adapted for ease ofreference. In all cases, the accepted IUPAC single letter or tripleletter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine with alanine atposition 226 is designated as “Thr226Ala” or “T226A”. Multiple mutationsare separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing mutations at positions 205 and 411substituting glycine (G) with arginine (R), and serine (S) withphenylalanine (F), respectively. In case that an amino acid substitutionin a particular position is specified where the position can be occupiedby different amino acids depending of the actual parent the originalamino acid is indicated as X or Xaa. For example “X226A” is intended tomean that the amino acid that occupies position 226 is substituted withA or Ala, independently of which amino acid occupies position 226 in theoriginal sequence (parent sequence).Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position*. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, new insertedamino acid. Accordingly the insertion of lysine after glycine atposition 195 is designated “Gly195GlyLys” or “G195GK”. Multipleinsertions of amino acids are designated [Original amino acid, position,original amino acid, new inserted amino acid #1, new inserted amino acid#2; etc.]. For example, the insertion of lysine and alanine afterglycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample the sequences would thus be:

Parent: Variant: 195 195 195a 195b G G-K-ADegenerate indications. For degenerate indications where an amino acidresidue identical to the existing amino acid residue is inserted,degeneracy in the nomenclature arises. For example, a glycine insertedafter the glycine in the above example would be indicated by “G195GG”.Given that an alanine were present at position 194, the same actualchange could just as well be indicated as “A194AG”:

Parent: Variant: Numbering I: 194 195 194 195 195a Sequence: A-G A-G-GNumbering II: 194 194a 195

Such instances will be apparent to the skilled person, and theindication “G195GG” and corresponding indications for this type ofinsertion is thus meant to comprise such equivalent degenerateindications.

If amino acid sequence segments are repeated in the parent polypeptideand/or in the variant, equivalent degenerate indications arise, alsowhen alterations other than insertions are listed such as deletionsand/or substitutions. For example, the deletion of two consecutive aminoacids “AG” in the sequence “AGAG” from position 194-97 may be written as“A194*+G195*” or “A196*+G197*”:

Parent: Variant: Numbering I: 194 195 196 197 194 195 Sequence: A-G-A-GA-G Numbering II: 196 197Multiple modifications. Variants comprising multiple modifications areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing modifications at positions 170 and 195substituting tyrosine and glutamic acid for arginine and glycine,respectively. Thus, “Tyr167Gly,Ala,Ser,Thr+Arg170Gly,Ala,Ser,Thr”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Gly+Arg170Ser”,

“Tyr167Gly+Arg170Thr”, “Tyr167Ala+Arg170Gly”, “Tyr167Ala+Arg170Ala”,

“Tyr167Ala+Arg170Ser”, “Tyr167Ala+Arg170Thr”, “Tyr167Ser+Arg170Gly”,

“Tyr167Ser+Arg170Ala”, “Tyr167Ser+Arg170Ser”, “Tyr167Ser+Arg170Thr”,

“Tyr167Thr+Arg170Gly”, “Tyr167Thr+Arg170Ala”, “Tyr167Thr+Arg170Ser”, and

“Tyr167Thr+Arg170Thr”.

This nomenclature is particularly relevant to modifications involvingsubstituting, inserting or deleting amino acid residues having specificcommon properties. Such modifications are referred to as conservativeamino acid modification(s). Examples of conservative modifications arewithin the group of basic amino acids (arginine, lysine and histidine),acidic amino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Amino acid modifications, which do not generally alterthe specific activity are known in the art and are described, forexample, by H. Neurath and R. L. Hill, 1979, In, The Proteins, AcademicPress, New York. The most commonly occurring exchanges are Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, andAsp/Gly as well as the reverse (Taylor, 1986, Journal of TheoreticalBiology 119: 205-218; compbio.dundee.ac.uk/papers/amas/amas3d.html).

Parent Alpha-Amylases

The parent alpha-amylase may in principle be any alpha-amylase for whichit is desired to prepare a variant having improved stability at low pH.Alpha-amylases are known derived from a vide selection of organismincluding bacteria, such as from species of the genus Bacillus, e.g.,Bacillus licheniformis; from species of fungi, such as Aspergillusoryzae (TAKA-amylase) or Aspergillus niger; from plants such as barleyand from mammals. The parent alpha-amylase may in principle be any suchalpha-amylase irrespective of the origin.

Termamyl-Like Alpha-Amylases

It is well known that a number of alpha-amylases produced by Bacillusspp. are highly homologous on the amino acid level as well as on thestructural level. For instance, the B. licheniformis alpha-amylasecomprising the amino acid sequence shown in SEQ ID NO: 4 (commerciallyavailable as Termamyl™) has been found to be about 81% homologous withthe B. amyloliquefaciens alpha-amylase comprising the amino acidsequence shown in SEQ ID NO: 6 and about 60% homologous with the B.stearothermophilus alpha-amylase comprising the amino acid sequenceshown in SEQ ID NO: 8. Further homologous alpha-amylases include analpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB12512, NCIB 12513 or DSM 9375, all of which are described in detail inWO 95/26397, and the SP #707 alpha-amylase described by Tsukamoto etal., 1988, Biochemical and Biophysical Research Communications 151:25-31.

Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like alpha-amylases are comprised inthe products sold under the following tradenames: Optitherm™ andTakatherm™ (available from Solvay); Maxamyl™ (available fromGist-brocades/Genencor), Spezym AA™ and Spezyme Delta AA™, Spezyme FRED(available from Genencor), and Keistase™ (available from Daiwa),Purastar™ ST 5000E, PURASTRA™ HPAM L (from Genencor Int.).

Because of the substantial homology found between these alpha-amylases,they are considered to belong to the same class of alpha-amylases,namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-likealpha-amylase” is intended to indicate an alpha-amylase, which at theamino acid level exhibits a substantial homology to Termamyl™, i.e., theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 4. In other words, a Termamyl-like alpha-amylase is analpha-amylase, which has the amino acid sequence shown in SEQ ID NO: 2,4, or 6, and the amino acid sequence shown in SEQ ID NO: 1 or 2 of WO95/26397 or in Tsukamoto et al. (1988), or the Bacillus flavothermusamylase, AMY1048 described in WO 2005/001064, or the alpha-amylase TS-22having the amino acid sequence of SEQ ID NO: 12; or the alpha-amylaseTS-23 having the amino acid sequence of SEQ ID NO: 13, described in J.Appl. Microbiology, 1997, 82: 325-334 (SWALL:q59222), or thealpha-amylase derived from Bacillus sp. KSM-AP1378 (FERM BP-3048) havingthe amino acid sequence of SEQ ID NO: 14, described in WO 97/00324, orthe alpha-amylase derived from Bacillus sp. A 7-7 having the amino acidsequence of SEQ ID NO: 15, described in WO 02/10356 or the Cytophagaalpha-amylase having the amino acid sequence of SEQ ID NO: 16, describedin Jeang et al., 2002, Appl. Environ. Microbiol. 68:3651-3654, or thealpha-amylase derived from Bacillus stearothermophilus (Spezyme Xtra),having the amino acid sequence of SEQ ID NO: 17; or the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811) or the alpha-amylases disclosed in WO 91/00353 and WO 94/18314 ori) which displays at least 60%, preferred at least 70%, more preferredat least 75%, even more preferred at least 80%, especially at least 85%,especially preferred at least 90%, even especially more preferred atleast 95% homology, more preferred at least 97%, more preferred at least99% with at least one of said amino acid sequences and/or ii) displaysimmunological cross-reactivity with an antibody raised against at leastone of said alpha-amylases, and/or iii) is encoded by a DNA sequencewhich hybridises to the DNA sequences encoding the above-specifiedalpha-amylases which are apparent from SEQ ID NOS: 1, 3, and 5 of thepresent application and SEQ ID NOS: 4 and 5 of WO 95/26397,respectively.

In a preferred embodiment the parent Termamyl-like alpha amylase is SEQID NO: 10 (SEQ ID NO: 2 of WO 95/26397), SEQ ID NO: 8 or SEQ ID NO: 2including any of [SEQ ID NO: 10]+R181*+G182*, [SEQ ID NO:10]+D183*+G184*; [SEQ ID NO: 10]+D183*+G184*+N195F; [SEQ ID NO:10]+D183*+G184*+M202L; [SEQ ID NO: 10]+D183*+G184*+N195F+M202L; [SEQ IDNO: 10]+D183*+G184*+R181Q; [SEQ ID NO:10]+D183*+G184*+R118K+N195F+R320K+R458K; [SEQ ID NO: 8]+I181*+G182*;[SEQ ID NO: 8]+I181*+G182*+N193F; [SEQ ID NO: 8]+I181*+G182*+M200L; [SEQID NO: 8]+I181*+G182*+N193F+M200L; [SEQ ID NO: 10]+D183*+G184*; [SEQ IDNO: 10]+D183*+G184*+N195F; [SEQ ID NO: 10]+D183*+G184*+M202L; [SEQ IDNO: 10]+D183*+G184*+N195F+M202L; [SEQ ID NO:10]+D183*+G184*+R118K+N195F+R320K+R458K. [SEQ ID NO:8]+I181*+G182*+N193F″ means the B. stearothermophilus alpha-amylasehaving SEQ ID NO: 8 has been mutated as follows: deletions in positionsI181 and G182 and a substitution from Asn (N) to Phe (F) in position193.

Parent Hybrid Alpha-Amylases

The parent alpha-amylase may be a hybrid alpha-amylase, i.e., analpha-amylase, which comprises a combination of partial amino acidsequences derived from at least two alpha-amylases.

The parent hybrid alpha-amylase may be one, which on the basis of aminoacid homology and/or immunological cross-reactivity and/or DNAhybridization (as defined above) can be determined to belong to theTermamyl-like alpha-amylase family. In this case, the hybridalpha-amylase is typically composed of at least one part of aTermamyl-like alpha-amylase and part(s) of one or more otheralpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination ofpartial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase, from which a partial amino acid sequence derives, may,e.g., be any of those specific Termamyl-like alpha-amylases referred toherein.

For instance, the parent alpha-amylase may comprise a C-terminal part ofan alpha-amylase derived from a strain of B. licheniformis, and aN-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus. Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 6 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 4, or b) an amino acid segment corresponding to the 68N-terminal amino acid residues of the B. stearothermophilusalpha-amylase having the amino acid sequence shown in SEQ ID NO: 8 andan amino acid segment corresponding to the 415 C-terminal amino acidresidues of the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 4.

In a preferred embodiment the parent Termamyl-like alpha-amylase is ahybrid Termamyl-like alpha-amylase identical to the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4, except that theN-terminal 35 amino acid residues (of the mature protein) is replacedwith the N-terminal 33 amino acid residues of the mature protein of theBacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 6.Said hybrid may further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4)referred to as LE174.

The non-Termamyl-like alpha-amylase may, e.g., be a fungalalpha-amylase, a mammalian or a plant alpha-amylase or a bacterialalpha-amylase (different from a Termamyl-like alpha-amylase). Specificexamples of such alpha-amylases include the Aspergillus oryzae TAKAalpha-amylase, the A. niger acid alpha-amylase, the Bacillus subtilisalpha-amylase, the porcine pancreatic alpha-amylase and a barleyalpha-amylase. All of these alpha-amylases have elucidated structures,which are markedly different from the structure of a typicalTermamyl-like alpha-amylase as referred to herein.

The fungal alpha-amylases mentioned above, i.e., derived from A. nigerand A. oryzae, are highly homologous on the amino acid level andgenerally considered to belong to the same family of alpha-amylases. Thefungal alpha-amylase derived from Aspergillus oryzae is commerciallyavailable under the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like alpha-amylase(variant of the invention) is referred to—in a conventional manner—byreference to modification (e.g., deletion or substitution) of specificamino acid residues in the amino acid sequence of a specificTermamyl-like alpha-amylase, it is to be understood that variants ofanother Termamyl-like alpha-amylase modified in the equivalentposition(s) (as determined from the best possible amino acid sequencealignment between the respective amino acid sequences) are encompassedthereby.

A preferred variant of the invention is one derived from a B.licheniformis alpha-amylase (as parent Termamyl-like alpha-amylase),e.g., one of those referred to above, such as the B. licheniformisalpha-amylase having the amino acid sequence shown in SEQ ID NO: 4.

Variants

The present invention relates to isolated variant alpha-amylases of aparent Termamyl-like alpha-amylase comprising two, three, four or fiveamino acid alterations in positions corresponding to positions in theparent alpha-amylase selected from the group consisting of 163, 188,205, 208 and 209; the alteration(s) are independently

(i) an insertion of an amino acid immediately downstream of theposition,

(ii) a deletion of the amino acid which occupies the position, and/or

(iii) a substitution of the amino acid which occupies the position,

wherein the variant has alpha-amylase activity and have reduced calciumsensitivity compared with the parent alpha-amylase, and wherein eachposition corresponds to a position of the amino acid sequence of theenzyme having the amino acid sequence of SEQ ID NO: 2.

The variant may be a natural or a non-natural variant, where naturalvariants should be understood as an alpha-amylase isolated from anaturally occurring organism that have not been the subject of humanmanipulation. A non-natural variant is a variant that have been modifiedfrom its natural counterpart, the parent alpha-amylase, by humanintervention using techniques such as mutation of a wild type organismand isolation of variant alpha-amylases, techniques involving isolationand manipulation of nucleic acids encoding a parent alpha-amylase orchemical synthesis of the variants of nucleic acids encoding them. Manysuch techniques are available in the art and the skilled person willappreciate that such techniques can be applied in the present invention.

The variants of the invention are generally isolated using at least oneseparation step, and the invention does therefore not apply to naturalenzymes in their natural environment.

The variants according to the invention have the benefit of being lesssensitive toward calcium depletion than their parent alpha-amylase, butat the same time they have maintained the performance properties of theparent alpha-amylase. Calcium sensitivity is manifested in the activityand/or stability of the particular alpha-amylase in calcium-depletedenvironments and/or under acidic conditions. Calcium-depletedenvironments occur in many known applications for alpha-amylases, suchas in the presence of strong chelators binding metal ions, in particularcalcium ions, e.g., in detergents, where it is common to include strongchelators because of the beneficial effect of the laundering process, orin conditions where plant material including natural chelators such asphytates or citrates is present. Such strong chelators will compete forthe calcium ions and will to some extend be able to deprivecalcium-sensitive alpha-amylases for the calcium ions bound in theirstructure with the consequence that the stability or activity of thecalcium sensitive alpha-amylase is reduced.

Acidic conditions may also affect the stability or activity ofcalcium-sensitive alpha-amylases. It is believed that low pH may lead toa protonation of the amino acid residues that coordinates the calciumions in sensitive alpha-amylases with the result that they no longer iscapable of binding the calcium and the result is a loss of stabilityand/or activity. As examples of applications where alpha-amylases areexposed to acidic conditions can be mentioned use of alpha-amylases asin treatment of digestive disorders such as disclosed in WO 2006/136161,and use in feed.

Thus, the variants of the invention have at least one of the properties:improved stability and/or activity in the presence of strong chelatorsand/or improved stability and/or activity at low pH, and it should beunderstood in this specification and claims that a variant havingreduced calcium sensitivity has improved stability and/or activity inthe presence of strong chelators and/or improved stability and/oractivity at low pH.

Chelator strength may be evaluated using methods known in the art suchas methods disclosed in Anal. Biochem. 314: 227-234 (2003), and JAOCS61(9): 1475-1478 (1984). As examples of strong chelators that may beused for such an assay can be mentioned EGTA (ethylene glycoltetraacetic acid), EDTA (ethylene diamine tetraacetic acid), DTPA(diethylene triamine pentaacetic acid), DTMPA (diethylenetriamine-penta-methylene phosphonic acid) and HEDP(1-hydroxyethan-1,1-diylbis(phosphonic acid)). The skilled person willbe able to select other strong chelators that may be used in determiningthe calcium sensitivity of an alpha-amylase.

In the present invention, the isolated variants of a parenttermamyl-like alpha-amylase comprise an alteration at two, three, fouror five positions, said positions corresponding to positions in theparent alpha-amylase selected from the group consisting of: 163, 188,205, 208 and 209 wherein the alteration(s) are independently

(i) an insertion of an amino acid immediately downstream of theposition,

(ii) a deletion of the amino acid which occupies the position, and/or

(iii) a substitution of the amino acid which occupies the position,

wherein the variant has alpha-amylase activity; and each positioncorresponds to a position of the amino acid sequence of the enzymehaving the amino acid sequence of SEQ ID NO: 2.

Preferably the variants comprises alterations at three positions, morepreferred four positions even more preferred five positions and mostpreferred six positions, said positions corresponding to positions inthe parent alpha-amylase selected from the group consisting of: 163,188, 205, 208 and 209 using SEQ ID NO: 2 for numbering.

The alterations may be selected among:

X163A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y, preferably X163Q or X163N;

X188A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y, preferably X188N;

X205A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y, preferably X205N;

X208A,C,D,E,F,G,H,I,K,L,N,P,Q,R,S,T,V,W,Y, preferably X208F or X208Y;and

X209A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y, preferably X2095 or X209N.

Preferred variants comprise the mutations D163Q+D188N+D209N,S or themutations D163Q,N+D188N+M208F+D209S+K242P+S244W.

The variants may further comprise additional alteration(s) in one ormore amino acid residues of the parent alpha-amylase.

Preferred further alterations include alterations where one or moreamino acids in the B-domain of the parent alpha-amylase are substitutedwith the corresponding amino acids of B. circulans alpha-amylase havingSEQ ID NO: 9 or Bacillus sp. KSM-K38 alpha-amylase SEQ ID NO: 11. Incase that a particular amino acid residue in the B-domain of a parentalpha-amylase is missing in SEQ ID NO: 9 or SEQ ID NO: lithe preferredalteration is a deletion of the particular amino acid. The skilledperson will appreciate that such a substitution of an amino acid residuein the B-domain of the parent alpha-amylase with the corresponding aminoacid in SEQ ID NO: 9 or SEQ ID NO: 11 is only relevant for positionswhere the two amino acid sequences differ. Consequently, the number ofpossible alterations in this group will vary depending on the particularparent alpha-amylase and the sequence identity between the parentalpha-amylase and the alpha-amylase having the sequence shown in SEQ IDNO: 9 or SEQ ID NO: 11.

The inventors have found that such further alterations provides forvariants having an even more reduced calcium sensitivity compared withthe parent alpha-amylase.

The B-domain of alpha-amylases is well known in the art and methods foridentifying B-domains known in the art can also be applied to thepresent invention. The B-domain of alpha-amylases can be determined bystructure analysis such as by using crystallographically techniques foridentification of the domain structure of a given alpha-amylase. Forexample, Machius et al., 1995, J. Mol. Biol. 4: 545-559 and Machius etal., 1998, Structure 6:281-292, identified the B-domain in B.licheniformis alpha-amylase as residues 104-204; Brzozowski et al.,2000, Biochemistry 39:9099-9017 identified the B-domain in a hybridalpha-amylase consisting of amino acids 1-300 of B. amyloliquefaciensalpha-amylase and amino acid residues 301-483 of B. licheniformisalpha-amylase as residues 104-205; Suvd et al., 2001, J. Biochem.129:461-468, identified the B-domain in B. stearopthermophilus alphaamylase and Nonaka et al. (2008) identified the B-domain of thealpha-amylase from Bacillus sp. KSM-K38.

An alternative method for determining the B-domain for a givenalpha-amylase is by sequence alignment of the amino acid sequence of thegiven alpha-amylase and an alpha-amylase for which the B-domain has beendetermined. The two sequences are aligned and the sequence in the givenalpha-amylase sequence that aligns with the B-domain sequence in thealpha-amylase for which the B-domain has been determined can for thepurpose of this invention be considered the B-domain for the givenalpha-amylase. This method is particular suitable for alpha-amylases forwhich the three-dimensional structure is not available. However, foralpha-amylases where the B-domain has been determined based on thethree-dimensional structure of the alpha-amylase, the B-domaindetermined by the latter method should preferentially be used in casethat the B-domain determined by alignment differs from the B-domaindetermined based on the structure.

The variants of the invention may even comprise further alterationsknown in the art to improve the performance of alpha-amylases. Forexample may oxidizable amino acid residues be substituted with anon-oxidizable amino acid residue in order to improve the stability ofthe enzyme under oxidizing conditions, e.g., in the presence of bleach,in accordance with the teachings of WO 94/18314 and WO 94/02597,incorporated herein by reference.

A two amino acid deletion may be introduced in positions correspondingto R181+G182 or T183+G184 in SEQ ID NO: 2 in accordance with theteachings of WO 96/23873, incorporated by reference.

Further beneficial substitutions that may be introduced into thevariants of the invention can be found in WO 99/23211, WO 01/66712, WO02/10355 and WO 2006/002643 all included by reference).

As examples of preferred further alterations can be mentionedD183*+G184*, G186A,Y,T, T193F, N195F, M202L,I,T,S,A, I206F,Y, V214I,S244A,D,E,N,Q,W, T452H,Y, G474R, G475R, wherein each positioncorresponds to a position of the amino acid sequence of the enzymehaving the amino acid sequence of SEQ ID NO: 2. The skilled person willappreciate that corresponding alteration can be identified and performedstarting from other parent alpha-amylases.

The number of amino acid substitutions in the variants of the presentinvention comprise preferably less than 60 substitutions, more preferredless than 55 substitutions, more preferred less than 50 substitutions,more preferred less than 45 substitutions, more preferred less than 40substitutions, more preferred less than 35 substitutions, more preferredless than 30 substitutions, more preferred less than 25 substitutions,more preferred less than 20 substitutions, more preferred less than 15substitutions, most preferred less than 10 substitutions.

The variants of the invention are preferably at least 70% identical totheir parent alpha-amylase, more preferred at least 75% identical totheir parent alpha-amylase; more preferred at least 80% identical totheir parent alpha-amylase, more preferred at least 85% identical totheir parent alpha-amylase more preferred at least 90% identical totheir parent alpha-amylase more preferred at least 95% identical totheir parent alpha-amylase, and most preferred at least 98% identical totheir parent alpha-amylase.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 4.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 6.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 8.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 10.

In another embodiment, the variant has at least 70%, e.g., at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, and at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 11.

In on preferred embodiment the parent alpha-amylase is the alpha-amylasehaving the sequence disclosed in SEQ ID NO: 2. In this embodimentpreferred substitutions according to the invention are selected among:D163Q,N, D188N, D205N, M208Y and D209N.

Alterations in the B-domain of SEQ ID NO: 2 include the alterations:A113E, M116V, V117F, R118K, A119V, V120I, N123D, N126D, N128T, Q129K,G133E, D134P, Y135F, T136E, A139G, D144T, N150D, T151Q, H152Y, N154S,R158N, W159S, Y160E, V165T, W167F, Q169A, S170R/K, R171E/G, K172E,L173R, N174*, N175T, R176G, 1177V, Y178F, K179R, F180I, R181A, D183E,G184N, A186K, W189E, E190N, T193D, N195F, Y203F, E212D, V214R, andN215R.

As examples of further alterations known to increase performance ofalpha-amylases can be mentioned: D183*+G184*, G186A,Y or T, T193F,N195F, M202X preferably L,I,T,S or A, I206F or Y, V214I, S244A,D,E,N,Qor W, T452H or Y, G474R and G475R.

Preferred variants according to this embodiment include:

D163Q+D188N+M208F+D209S+K242P+S244W;

D163N+R181A+G182A+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;

N128W+D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;

D163N+R181A+G183N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+H408W+N409D+D432N+A434P;

D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+N409D+D432N+A434P;

D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W

D183*+G184*+R118K+N195F+R320K+R458K+D163Q+D188N+M208F+D209S+K242P+S244W;

D183*+G184*+R118K+N195F+R320K+R458K+D163N+R181A+G182A+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;

D183*+G184*+R118K+N195F+R320K+R458K+N128W+D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;

D183*+G184*+R118K+N195F+R320K+R458K+D163N+R181A+G183N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+H408W+N409D+D432N+A434P;

D183*+G184*+R118K+N195F+R320K+R458K;

D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;

D183*+G184*+R118K+N195F+R320K+R458K+D163N+R181A+G182A+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;

D188N+D209S;

D163N+D188N+D209S;

D163N+D188N+D205N+D209S;

D163N+D188N+D205N+M208F+D209S;

D207N+D209S;

D163N+D207N+D209S;

D163N+D188N+D207N+D209S;

D163N+D188N+D199N+D207N+D209S;

D163N+D188N+D199N+D205N+D207N+D209S;

D163N+D188N+D199N+D205N+M208F+D207N+D209S;

D163N+R181A+G182N+G186N+D188N+D205N+D209S;

D163N+R181A+G182N+K185T+G186N+D188N+D205N+D209S;

D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S;

D163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209S;

D163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209S; and

D163N+R181A+G182N+K185T+G186N+D188N+D199N+D205N+M208F+D207N+D209S.

In another preferred embodiment the parent alpha-amylase is SP722 havingthe sequence disclosed in SEQ ID NO: 10. In this embodiment preferredsubstitutions according to the invention are selected among: D163Q,N,D188N, D205N, M208Y and D209N.

Alterations in the B-domain of SEQ ID NO: 10 include the alterations:A113E, N116V, V117F, L118K, A119V, V120I, N123D, N126D, N128T, Q129K,G133E, D134P, Y135F, T136E, A139G, D144T, N150D, T151Q, D154S, R158N,W159S, Y160E, V165T, W167F, Q169A, S170R or K, R171E or G, Q172E, F173R,Q174*, N175T, R176G, I177V, Y178F, K179R, F180I, R181A, D183E, G184N,A186K, W189E, E190N, S193D, N195F, Y203F, V206I, E212D, V214R, andN215R.

As examples of further alterations known to increase performance ofalpha-amylases can be mentioned: D183*+G184*, A186Y or T, S193F, N195F,M202X, preferably L,I,T,S or A, I206L or F, V214I, S244A,E or Q, H452Y,G474R and G475R.

As examples or preferred variants according to this embodiment can bementioned:

D188N+D209S;

D163N+D188N+D209S;

D163N+D188N+D205N+D209S;

D163N+D188N+D205N+M208F+D209S;

D207N+D209S;

D163N+D207N+D209S;

D163N+D188N+D207N+D209S;

D163N+D188N+D199N+D207N+D209S;

D163N+D188N+D199N+D205N+D207N+D209S;

D163N+D188N+D199N+D205N+M208F+D207N+D209S;

D163N+R181A+G182N+G186N+D188N+D205N+D209S;

D163N+R181A+G182N+K185T+A186N+D188N+D205N+D209S;

D163N+R181A+G182N+K185T+A186N+D188N+D205N+M208F+D209S;

D163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209S;

D163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209S; and

D163N+R181A+G182N+K185T+A186N+D188N+D199N+D205N+M208F+D207N+D209S.

In a further preferred embodiment the parent alpha-amylase is the B.stearothermophilus alpha-amylase having the sequence disclosed in SEQ IDNO: 8. In this embodiment preferred substitutions are selected among:D162Q,N, D186N, D203N, M206Y and D207N.

Alterations in the B-domain of SEQ ID NO: 8 where an amino acid issubstituted with the corresponding amino acid from the alpha-amylasehaving the sequence SEQ ID NO: 9 or deletions in case that nocorresponding amino acid exists include the alterations: D105N, G108A,G112E, W115V, V116F, D117K, A118V, V119I, N122D, S124N, N127T, Q128K,G132E, T133P, Y134F, Q135E, A138G, D143T, N149D, T150Q, R157N, W158S,Y159E, V164T, W166F, E168A, S169K, R170G, K171E, L172R, S173T, R174G,I175V, Y176F, K177R, F178I, R179A, I181E, G182N, A184K, W187E, E188N,T191 D, Y201F, L204I and, E210D.

As examples of further alterations known to increase performance ofalpha-amylases are: I181*+G182*, A184Y/T, N191F, N193F, M200X preferablyL,I,T,S or A, L204F, M206Y, V212I, S242A,D,E,N or Q, H296Y, G474R andG475R.

As examples or preferred variants according to this embodiment can bementioned:

D207N+D186N;

D207N+D186N+D162N;

D207N+D186N+D162N+D203N;

D207N+D186N+D162N+D203N+M206Y;

D207N+D186N+D162N+D203N+M206Y+D105N;

D207N+A184K+W187E;

D162N+A184K+D186N+D207N;

D207N+A184K+W187E+D186N;

D207N+A184K+W187E+D186N+D162N;

D207N+A184K+W187E+D186N+D162N+D203N;

D207N+A184K+W187E+D186N+D162N+D203N+M206Y; and

D207N+A184K+W187E+D186N+D162N+D203N+M206Y+D105N.

Other preferred parent alpha-amylases includes: SEQ ID NO: 2 in WO2005/001064, B. licheniformis alpha-amylase having the sequence SEQ IDNO: 4, B. amyloliquefaciens alpha-amylase having the sequence SEQ ID NO:6, alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289,NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detailin WO 95/26397, and the #707 alpha-amylase described by Tsukamoto etal., 1988, Biochemical and Biophysical Research Communications, 151:25-31 and the alpha-amylase derived from KSM-Ap1378 and described in WO94/26881.

Nucleotide Sequences

Cloning a DNA Sequence Encoding an Alpha-Amylase

The DNA sequence encoding a parent alpha-amylase may be isolated fromany cell or microorganism producing the alpha-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the alpha-amylase to be studied. Then,if the amino acid sequence of the alpha-amylase is known, homologous,labelled oligonucleotide probes may be synthesized and used to identifyalpha-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to a known alpha-amylase gene could beused as a probe to identify alpha-amylase-encoding clones, usinghybridization and washing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase, thereby allowingclones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or Saikiet al. (1988).

Site-Directed Mutagenesis

Once an alpha-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 disclose the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNAsequences is described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Homology (Identity)

The homology may be determined as the degree of identity between the twosequences indicating a derivation of the first sequence from the second.The homology may suitably be determined by means of computer programsknown in the art such as GAP provided in the GCG program package(described above). Thus, Gap GCGv8 may be used with the default scoringmatrix for identity and the following default parameters: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3, respectively fornucleic acidic sequence comparison, and GAP creation penalty of 3.0 andGAP extension penalty of 0.1, respectively, for protein sequencecomparison. GAP uses the method of Needleman and Wunsch, 1970, J. Mol.Biol. 48: 443-453, to make alignments and to calculate the identity.

A structural alignment between Termamyl and a Termamyl-likealpha-amylase may be used to identify equivalent/corresponding positionsin other Termamyl-like alpha-amylases. One method of obtaining saidstructural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., 1987, FEBS Letters 224: 149-155) and reverse threading (Huber etal., 1998, Protein Science 7(1): 142-149). Property ii) of thealpha-amylase, i.e., the immunological cross reactivity, may be assayedusing an antibody raised against, or reactive with, at least one epitopeof the relevant Termamyl-like alpha-amylase. The antibody, which mayeither be monoclonal or polyclonal, may be produced by methods known inthe art, e.g., as described by Hudson et al., Practical Immunology,Third edition (1989), Blackwell Scientific Publications. Theimmunological cross-reactivity may be determined using assays known inthe art, examples of which are Western Blotting or radialimmunodiffusion assay, e.g., as described by Hudson et al., 1989. Inthis respect, immunological cross-reactivity between the alpha-amylaseshaving the amino acid sequences SEQ ID NOS: 2, 4, 6, or 8, respectively,have been found.

Hybridization

The oligonucleotide probe used in the characterization of theTermamyl-like alpha-amylase may suitably be prepared on the basis of thefull or partial nucleotide or amino acid sequence of the alpha-amylasein question.

Suitable conditions for testing hybridization involve presoaking in5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridization inthe same solution supplemented with 100 mM ATP for 18 hours at ˜40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at ˜75° C. (very high stringency). More details about thehybridization method can be found in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate analpha-amylase, which is encoded by a DNA sequence of synthetic and/orcDNA origin and which has the identifying characteristics of thealpha-amylase in question. The term is also intended to indicate thatthe parent alpha-amylase may be a variant of a naturally occurringalpha-amylase, i.e., a variant, which is the result of a modification(insertion, substitution, deletion) of one or more amino acid residuesof the naturally occurring alpha-amylase.

Production of Variant Alpha-Amylases

The variant alpha-amylases of the invention may be produced usingmethods well known in the area. Generally, DNA sequences encoding theparent alpha-amylase is provided and the desired alteration is generatedin the nucleic acid sequence using techniques known in the art.

The generated DNA sequence encoding the desired variant alpha-amylase ofthe invention is provided with suitable regulatory sequences, such aspromoter, terminator, activation sites, ribosome binding sites,polyadenylation sites etc. and introduced into a suitable host cell.Finally the host cell comprising said DNA is grown under conditionsleading to expression of the variant alpha-amylase according to theinvention.

All these techniques are known in the art and it is within the skills ofthe average practitioner within the field to prescribe a suitable methodfor producing a given variant alpha-amylase of the invention usingtechniques disclosed in well known text books such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989.

Further teachings regarding preparation of variant alpha-amylases can befound in WO 2006/002643, which is incorporated by reference, and theskilled person will appreciate that this teaching also applies to thepresent invention.

Compositions

The present invention also relates to compositions comprising analpha-amylase variant and at least one additional enzyme. The additionalenzyme(s) may be selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase),glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease, pullulanase, and/or other enzymes useful in acommercial process in conjunction with an alpha-amylase. The additionalenzyme may also be a second alpha-amylase. Such enzymes are known in theart in starch processing, sugar conversion, fermentations for alcoholand other useful end-products, commercial detergents and cleaning aids,stain removal, fabric treatment or desizing, and the like.

Methods of Using the Alpha-Amylase Variants—Industrial Applications

The variants of the present invention possess valuable propertiesallowing for a variety of industrial applications. In particular, thevariants may be used in detergents, in particular laundry detergentcompositions and dishwashing detergent compositions, hard surfacecleaning compositions, and for desizing textiles, fabrics or garments,production of pulp and paper, beer making, ethanol production, andstarch conversion processes.

The alpha-amylase variants may be used for starch processes, inparticular starch conversion, especially liquefaction of starch (see,e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, andWO 96/28567, which are all hereby incorporated by reference). Alsocontemplated are compositions for starch conversion purposes, which maybeside the variant of the invention also comprise an AMG, pullulanase,and other alpha-amylases.

Further, the variants are particularly useful in the production ofsweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which ishereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The variants may also be used for desizing of textiles, fabrics, andgarments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP119920, which are incorporated herein by reference), beer making orbrewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps: milling, liquefaction, saccharification,and fermentation.

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by analpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are)added. The liquefaction process is carried out at 85° C. for 1-2 hours.The pH is generally between 5.5 and 6.2. In order to ensure optimalenzyme stability under these conditions, 1 mM of calcium is added (toprovide about 40 ppm free calcium ions). After such treatment, theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g.,105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C., which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., 4.5. In a simultaneous saccharification and fermentation(SSF) process, there is no holding stage for saccharification, rather,the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof a glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list are only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material. The fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material and water. In one embodiment aprocess of the invention includes saccharifying (e.g., milled)starch-containing material, e.g., granular starch, below the initialgelatinization temperature, preferably in the presence of alpha-amylaseand/or carbohydrate-source generating enzyme(s) to produce sugars thatcan be fermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn. Accordingly, in the first aspectthe invention relates to processes for producing fermentation productsfrom starch-containing material comprising simultaneously saccharifyingand fermenting starch-containing material using a carbohydrate-sourcegenerating enzyme and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material.In an embodiment a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast, e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55, e.g.,25-45 and 30-40, w/w % dry solids (DS) of starch-containing material maybe prepared. The slurry may include water and/or process waters, such asstillage (backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process carried at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %, such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase variant, or;

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is alsoused in the liquefaction step. In an embodiment a protease, such as anacid fungal protease or a metallo protease is added before, duringand/or after liquefaction. In an embodiment the metalloprotease isderived from a strain of Thermoascus, e.g., a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In anembodiment the carbohydrate-source generating enzyme is a glucoamylasederived from a strain of Aspergillus, e.g., Aspergillus niger orAspergillus awamori, a strain of Talaromyces, especially Talaromycesemersonii; or a strain of Athelia, especially Athelia rolfsii; a strainof Trametes, e.g., Trametes cingulata; a strain of the genusPachykytospora, e.g., a strain of Pachykytospora papyracea; or a strainof the genus Leucopaxillus, e.g., Leucopaxillus giganteus; or a strainof the genus Peniophora, e.g., a strain of the species Peniophorarufomarginata; or a mixture thereof. Saccharification step (b) andfermentation step (c) may be carried out either sequentially orsimultaneously. A pullulanase and/or metalloprotease may be added duringsaccharification and/or fermentation when the process is carried out asa sequential saccharification and fermentation process and before orduring fermentation when steps (b) and (c) are carried outsimultaneously (SSF process). The pullulanase and/or metalloprotease mayalso advantageously be added before liquefaction (pre-liquefactiontreatment), i.e., before or during step (a), and/or after liquefaction(post liquefaction treatment), i.e., after step (a). The pullulanase ismost advantageously added before or during liquefaction, i.e., before orduring step (a). The fermentation product, such as especially ethanol,may optionally be recovered after fermentation, e.g., by distillation.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces cerevisiae. In a particular embodiment, the process of theinvention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In an embodiment the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55 w/w % dry solids(DS), e.g., 25-45 and 30-40 w/w % dry solids (DS) of starch-containingmaterial. The slurry is heated to above the gelatinization temperatureand an alpha-amylase variant may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to alpha-amylase in step(a). Liquefaction may in an embodiment be carried out as a three-stephot slurry process. The slurry is heated to between 60-95° C.,preferably between 70-90° C., such as preferably between 80-85° C. at pH4-6, preferably 4.5-5.5, and alpha-amylase variant, optionally togetherwith a pullulanase and/or protease, preferably metalloprotease, areadded to initiate liquefaction (thinning). In an embodiment the slurrymay then be jet-cooked at a temperature between 95-140° C., preferably100-135° C., such as 105-125° C., for about 1-15 minutes, preferably forabout 3-10 minutes, especially around about 5 minutes. The slurry iscooled to 60-95° C. and more alpha-amylase variant and optionallypullulanase variant and/or protease, preferably metalloprotease, is(are)added to finalize hydrolysis (secondary liquefaction). The liquefactionprocess is usually carried out at pH 4.0-6, in particular at a pH from4.5 to 5.5. Saccharification step (b) may be carried out usingconditions well known in the art. For instance, a full saccharificationprocess may last up to from about 24 to about 72 hours, however, it iscommon only to do a pre-saccharification of typically 40-90 minutes at atemperature between 30-65° C., typically about 60° C., followed bycomplete saccharification during fermentation in a simultaneoussaccharification and fermentation process (SSF process).Saccharification is typically carried out at temperatures from 20-75°C., preferably from 40-70° C., typically around 60° C., and at a pHbetween 4 and 5, normally at about pH 4.5. The most widely used processto produce a fermentation product, especially ethanol, is a simultaneoussaccharification and fermentation (SSF) process, in which there is noholding stage for the saccharification, meaning that a fermentingorganism, such as yeast, and enzyme(s), may be added together. SSF maytypically be carried out at a temperature from 25° C. to 40° C., such asfrom 28° C. to 35° C., such as from 30° C. to 34° C., preferably aroundabout 32° C. In an embodiment fermentation is ongoing for 6 to 120hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylase variants may also be used in a beer-making process andsimilar fermentations; the alpha-amylases will typically be added duringthe mashing process. The process is substantially similar to themilling, liquefaction, saccharification, and fermentation processesdescribed above.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and5.2 depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to a phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially-available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch maybe further hydrolyzed at atemperature ranging from about 75° C. to 95° C. (e.g., 80° C. to 90° C.and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, andpH 5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylase variants may also be used in the production oflignocellulosic materials, such as pulp, paper and cardboard, fromstarch reinforced waste paper and cardboard, especially where re-pulpingoccurs at pH above 7 and where amylases facilitate the disintegration ofthe waste material through degradation of the reinforcing starch. Thealpha-amylase variants are especially useful in a process for producinga papermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylase variants may also be useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealpha-amylase variants it is possible to modify the starch in thepresence of the filler thus allowing for a simpler integrated process.

Desizinq of Textiles, Fabrics and Garments

The alpha-amylase variants may also be very useful in textile, fabric orgarment desizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yarns during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing process is sometimes combinedwith the scouring and bleaching steps. In such cases, non-enzymaticauxiliaries such as alkali or oxidation agents are typically used tobreak down the starch, because traditional alpha-amylases are not verycompatible with high pH levels and bleaching agents. The non-enzymaticbreakdown of the starch size leads to some fiber damage because of therather aggressive chemicals used. Accordingly, it would be desirable touse the alpha-amylase variants as they have an improved performance inalkaline solutions. The alpha-amylase variants may be used alone or incombination with a cellulase when desizing cellulose-containing fabricor textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in, e.g., WO 95/21247, U.S. Pat.No. 4,643,736, EP 119920, which are hereby incorporated by reference.

Cleaning Processes and Detergent Compositions

The alpha-amylase variants may be added as a component of a detergentcomposition for various cleaning or washing processes, including laundryand dishwashing. For example, the variants may be used in the detergentcompositions described in WO 96/23874 and WO 97/07202.

The alpha-amylase variants may be incorporated in detergents atconventionally employed concentrations. For example, a variant of theinvention may be incorporated in an amount corresponding to 0.00001-10mg (calculated as pure, active enzyme protein) of alpha-amylase perliter of wash/dishwash liquor using conventional dosing levels ofdetergent.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition, including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more otherenzymes, such as a lipase, peroxidase, protease, another amylolyticenzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase,CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes,Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols, fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleiclacrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19708 and WO92/19709.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.055 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor. One ormore of the variant enzymes described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

This disclosure includes further detail in the following examples, whichare not in any way intended to limit the scope of what is claimed. Thefollowing examples are thus offered to illustrate, but not to limit whatis claimed.

EXAMPLES

Materials

Enzymes

SP722: SEQ ID NO: 10, available from Novozymes, and disclosed in WO95/26397.

AA560: SEQ ID NO: 2; disclosed in WO 00/60060 and available fromNovozymes A/S; disclosed in Danish patent application no. PA 1999 00490;deposited on Jan. 25, 1999 at DSMZ and assigned the DSMZ no. 12649.

Bacillus subtilis SHA273: see WO 95/10603

Methods

General Molecular Biology Methods

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989); Ausubel et al. (1995); Harwood and Cutting (1990)).

Fermentation of Alpha-Amylases and Variants

Fermentation may be performed by methods well known in the art or asfollows.

A B. subtilis strain harboring the relevant expression plasmid isstreaked on a LB-agar plate with a relevant antibiotic, and grownovernight at 37° C.

The colonies are transferred to 100 ml BPX media supplemented with arelevant antibiotic (for instance 10 mg/l chloroamphinicol) in a 500 mlshaking flask.

Composition of BPX Medium:

Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodiumcaseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12H₂O 9 g/l Pluronic ™0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 4 to 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on an UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM acetate pH 5.5. The UF-filtrate is applied onan S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3 M NaCl over 6 column volumes. The fractions, which containthe activity (measured by the Phadebas assay) are pooled, pH wasadjusted to pH 7.5 and remaining color was removed by a treatment with0.5% w/vol. active coal in 5 minutes.

Determination of Residual Activity at pH 4.0

Reagents etc.

-   Buffer: 50 mM Citrate, pH 4, 0.05% Triton-X100

Prepare 500 mM stock solution.

-   Stability buffer: 50 mM Carbonate buffer (NaHCO₃), pH 8, 1 mM CaCl₂,    0.05% Triton-X100    Sample Preparation

Samples are centrifuged at 20,000 rpm for 2 minutes. If necessary thesamples may be diluted in a stability buffer.

Incubation

100 microliters prepared sample

Ad 1000 microliters buffer

Incubate at 35° C. and withdraw 20 microliters aliquots in a 200microliters cold stability buffer after 0, 20 and 48 hours. These can bestored on ice for later activity determination.

Assay

Measure the residual activity using the Phabedas assay, see protocolbelow.

Measurement of the Calcium- and pH-Dependent Stability

Normally industrial liquefaction processes runs using pH 6.0-6.2 asliquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95° C.-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

1. pH lower than pH 6.2 and/or

2. free calcium levels lower than 40 ppm.

Two different methods can be used to measure the alterations instability obtained by the different substitutions in the alpha-amylasein question:

Method 1. One assay which measures the stability at reduced pH, pH 5.0,in the presence of 5 ppm free calcium.

10 micro g of the variant are incubated under the following conditions:A 0.1 M acetate solution, pH adjusted to pH 5.0, containing 5 ppmcalcium and 5% w/w common corn starch (free of calcium). Incubation ismade in a water bath at 95° C. for 30 minutes.

Method 2. One assay, which measure the stability in the absence of freecalcium and where the pH is maintained at pH 6.0. This assay measuresthe decrease in calcium sensitivity:

10 micro g of the variant were incubated under the following conditions:A 0.1 M acetate solution, pH adjusted to pH 6.0, containing 5% w/wcommon corn starch (free of calcium). Incubation was made in a waterbath at 95° C. for 30 minutes.

Assays for Alpha-Amylase Activity

1. Phadebas Assay

Alpha-amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin ×ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

Alpha-amylase activity is determined by a method employing the PNP-G7substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim(cat. no. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is addedto 5 ml buffer (BM1442309). To prepare the alpha-glucosidase one bottleof alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).The working solution is made by mixing 5 ml alpha-Glucosidase solutionwith 1 ml substrate.

The assay is performed by transforming 20 microliters enzyme solution toa 96 well microtitre plate and incubating at 25° C. 200 microlitersworking solution, 25° C. is added. The solution is mixed andpre-incubated 1 minute and absorption is measured every 15 sec. over 3minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of thealpha-amylase in question under the given set of conditions.

3. Enzchek® Amylase Activity Assay

Alpha-amylase activity may also be determined by a method employing theEnzChek® substrate. The substrate in the EnzChek® Ultra Amylase AssayKit (E33651, Invitrogen, La Jolla, Calif., USA) is a corn starchderivative, DQ™ starch, which is corn starch labeled with BODIPY® FL dyeto such a degree that fluorescence is quenched.

One vial containing approx. 1 mg lyophilized substrate is dissolved in100 microliters of 50 mM sodium acetate (pH 4.0). The vial is vortexedfor 20 seconds and left at room temperature, in the dark, withoccasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (w/v) TRITON® X100, 0.12 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The substrate working solution is prepared by diluting10-fold in residual activity buffer (100 mM acetate, 0.01% (w/v) TRITON®X100, 0.12 mM CaCl₂, pH 5.5) giving a substrate concentration of 100micrograms/ml. Immediately after incubation the enzyme is diluted to aconcentration of 20 ng enzyme protein/mL in 100 mM acetate, 0.01% (W/v)TRITON® X100, 0.12 mM CaCl₂, pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Example 1 Preparation of Variants

Using the parent alpha-amylaseAA560+delta(D183+G184)+N195F+R118K+R320K+R458K (disclosed in WO01/66712) following variants were constructed:

-   1. D163Q+D188N+M208F+D209S+K242P+S244W;-   2.    D163N+R181A+G182A+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;-   3.    N128W+D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;-   4.    D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+N409D+D432N+A434P;-   5. D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W

Example 2 Measurement of Residual Activity at pH 4.0

The residual activity at pH 4.0 was measured for the parent amylase and5 variants prepared in example 1. All measurements were done as doubledeterminations. The results are expressed in percent of the initialactivity.

The following results were obtained demonstrating the improved stabilityof the variants of the invention at pH 4.0:

0 hours 20 hours 48 hours Parent alpha-amylase 100% 35% 25% variant 1100% 65% 34% variant 2 100% 80% 70% variant 3 100% 87% 85% variant 4100% 88% 84% variant 5 100% 97% 96%

Example 3 Residual Activity after Incubation with Strong Chelators

The parent alpha-amylase and variant 5 described in example 1 wasincubated in the presence of strong chelators.

For incubation with chelators following mixtures were prepared:

100 microliters 250 mM EDTA or 100 microliters 10% DTPA

100 microliters enzyme preparation

Ad 1000 microliters with buffer.

Samples were incubated at 35° C. for 18 hours and the activity wasdetermined using the PNP-G7 method described above.

Parent Variant 5 DTPA 6% 42% EDTA 15% 79%

The results clearly show that the variant of the invention isconsiderably more resistant to the presence of strong chelators than theparent alpha-amylase.

Example 4 Measurement of Residual Activity at pH 3.0

Variant 5 and the parent alpha-amylase described in example 1 wereincubated at pH 3.0 at 35° C. for 18 hours and the residual activity wasdetermined using the PNP-G7 assay described above.

Parent Variant 5 pH 3 1% 79%

The results show the variant of the invention also 0 has improvedstability at pH 3. compared with the parent alpha-amylase.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention is further defined in the following paragraphs:

Paragraph 1. An isolated variant alpha-amylase, comprising two or morealterations at positions corresponding to positions 163, 188, 205, 208,and 209 of the mature polypeptide of SEQ ID NO: 2, wherein

(a) the variant has a sequence identity to any of SEQ ID NOS: 2, 4, 6,8, 10, 11, 12, 13, 14, 15, 16, and 17 of at least 70% and less than100%.

(b) each alteration is independently a substitution, deletion orinsertion; and

(c) the variant has alpha-amylase activity.

Paragraph 2. The variant of paragraph 1, which has at least 80% sequenceidentity to sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 11,12, 13, 14, 15, 16, and 17.

Paragraph 3. The variant of paragraph 1, which at least 90% sequenceidentity to sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 11,12, 13, 14, 15, 16, and 17.

Paragraph 4. The variant of paragraph 1, which at least 95% sequenceidentity to sequence identity to any of SEQ ID NOS: 2, 4, 6, 8, 10, 11,12, 13, 14, 15, 16, and 17.

Paragraph 5. The variant of any of paragraphs 1-4, which wherein thealterations at positions 163, 188, 205, 208, and 209 are substitutions.

Paragraph 6. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163 and 188.

Paragraph 7. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163 and 205.

Paragraph 8. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163 and 208.

Paragraph 9. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163 and 209.

Paragraph 10. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188 and 205.

Paragraph 11. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188 and 208.

Paragraph 12. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188 and 209.

Paragraph 13. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 205 and 208.

Paragraph 14. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 205 and 209.

Paragraph 15. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 208 and 209.

Paragraph 16. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, and205.

Paragraph 17. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, and208.

Paragraph 18. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, and209.

Paragraph 19. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188, 205, and208.

Paragraph 20. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188, 205, and209.

Paragraph 21. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 205, 208, and209.

Paragraph 22. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, 205,and 208.

Paragraph 23. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, 205,and 209.

Paragraph 24. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 188, 205, 208,and 209.

Paragraph 25. The variant of any of paragraphs 1-4, which comprises asubstitution at the positions corresponding to positions 163, 188, 205,208, and 209.

Paragraph 26. The variant of any of paragraphs 1-25, wherein thealterations are selected among: X163Q,N, X188N, X205N, X208Y andX209N,S.

Paragraph 27. The variant of any of paragraphs 1-26, further comprisingone or more alterations selected from the group consisting of a deletionat a position corresponding to positions 183 and 184 and a substitutionat a position corresponding to the positions selected from the groupconsisting of 186, 193, 195, 202, 206, 214, 244, 452, 474 and 475Paragraph 28. The variant of any of paragraphs 1-27, which furthercomprises one or more alterations selected from the group ofX181*+X182*, X182*+X183*, X183*+X184*, X185K, X167W, X202L/I/T, X203Y,X167W+X168E+X169E+X170R, X51T+X109G+X203Y, X109G+X203Y, X189W,X189W+x190E+x193T, X190E, X193T, X303K,X303K+x305R+x306D+X409N+X432N+X434D, X305R, X306D, X409N, X432N, X434D.Paragraph 29. The variant of any of paragraphs 1-28, which furthercomprises one or more alterations selected from the group consisting ofA113E, N116V, V117F,L118K, A119V,V120I,N123D, N126D, N128T, Q129K,G133E, D134P, Y135F, T136E, A139G, D144T, N150D, T151Q, D154S, R158N,W159S, Y160E, V165T, W167F, Q169A, S170K, R171G, Q172*, F173E, Q174R,N175T, R176G, I177V, Y178F, K179R, F180I, R181A, D183E, G184N, A186K,W189E, E190N, S193T/D, N195F, Y203F,V206I, E212D, V214R, and N215R.Paragraph 30. The variant of any of paragraphs 1-29, which furthercomprises one or more alterations selected from the group consisting ofD183*+G184*, G186A,Y,T, T193F, N195F, M202L,I,T,S,A, I206F,Y, V214I,S244A,D,E,N,Q,W, T452HY, G474R, G475R.Paragraph 31. The variant of any of paragraphs 1-30 selected from thegroup consisting of:D188N+D209S;D163N+D188N+D209S;D163N+D188N+D205N+D209S;D163N+D188N+D205N+M208F+D209S;D207N+D209S;D163N+D207N+D209S;D163N+D188N+D207N+D209S;D163N+D188N+D199N+D207N+D209S;D163N+D188N+D199N+D205N+D207N+D209S;D163N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+G186N+D188N+D205N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S;D163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+N409D+D432N+A434P;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+H408W+N409D+D432N+A434P;N128W+D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;andD163Q+D188N+M208F+D209S+K242P+S244W.Paragraph 32. The variant of any of paragraphs 1-31 selected from thegroup consisting of:D188N+D209SD163N+D188N+D209SD163N+D188N+D205N+D209SD163N+D188N+D205N+M208F+D209SD207N+D209SD163N+D207N+D209SD163N+D188N+D207N+D209SD163N+D188N+D199N+D207N+D209SD163N+D188N+D199N+D205N+D207N+D209SD163N+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+G186N+D188N+D205N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D205N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D205N+M208F+D209SD163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D199N+D205N+M208F+D207N+D209S.Paragraph 33. The variant of any of paragraphs 1-32 selected from thegroup consisting of:D207N+D186ND207N+D186N+D162ND207N+D186N+D162N+D203ND207N+D186N+D162N+D203N+M206YD207N+D186N+D162N+D203N+M206Y+D105ND207N+A184K+T187ED207N+A184K+T187E+D186ND207N+A184K+T187E+D186N+D162ND207N+A184K+T187E+D186N+D162N+D203ND207N+A184K+T187E+D186N+D162N+D203N+M206YD207N+A184K+T187E+D186N+D162N+D203N+M206Y+D105N.Paragraph 34. A detergent composition comprising a variant of any ofparagraphs 1-33 and a surfactant.Paragraph 35. A composition comprising a variant of any of paragraphs1-33 and one or more enzymes selected from the group consisting ofbeta-amylase, cellulase (beta-glucosidase, cellobiohydrolase, andendoglucanase) glucoamylase, hemicellulase (e.g., xylanase), isoamylase,isomerase, lipase, phytase, protease, and pullulanase.Paragraph 36. Use of a variant of any of paragraphs 1-33 for washingand/or dishwashing.Paragraph 37. Use of a variant of any of paragraphs 1-33 for desizing atextile.Paragraph 38. Use of a variant of any of paragraphs 1-33 for producing abaked product.Paragraph 39. Use of a variant of any of paragraphs 1-33 for liquefyinga starch-containing material.Paragraph 40. A method of producing liquefied starch, comprisingliquefying a starch-containing material with a variant of any ofparagraphs 1-33Paragraph 41. A process of producing a fermentation product, comprising

(a) liquefying a starch-containing material with a variant of any ofparagraphs 1-33 to produce a liquefied mash;

(b) saccharifying the liquefied mash to produce fermentable sugars; and

(c) fermenting the fermentable sugars in the presence of a fermentingorganism.

Paragraph 42. The process of paragraph 41 wherein the starch-containingmaterial is liquefied with the variant and a pullulanase, e.g., a GH57pullulanase.

Paragraph 43. The process of paragraph 42 wherein the pullulanase isobtained from a strain of Thermococcus, including Thermococcus sp. AM4,Thermococcus sp. HJ21, Thermococcus barophilus, Thermococcusgammatolerans, Thermococcus hydrothermalis; Thermococcus kodakarensis,Thermococcus litoralis, and Thermococcus onnurineus; or from a strain ofPyrococcus, such as Pyrococcus abyssi and Pyrococcus furiosus.Paragraph 44. The process of any of paragraphs 41-43 further comprisingadding a protease, such as an acid fungal protease or a metalloproteasebefore, during and/or after liquefaction.Paragraph 45. The process of paragraph 44, wherein the metalloproteaseis obtained from a strain of Thermoascus, preferably a strain ofThermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No.0670.Paragraph 46. A process of producing a fermentation product, comprisingcontacting a starch substrate with a variant of any of paragraphs 1-33,a glucoamylase, and a fermenting organism.Paragraph 47. The process of any of paragraphs 41-46, wherein thefermentation product is selected from the group consisting of alcohol(e.g., ethanol and butanol), organic acids (e.g., succinic acid andlactic acid), sugar alcohols (e.g., glycerol), ascorbic acidintermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).Paragraph 48. An isolated polynucleotide encoding the variant of any ofparagraphs 1-33.Paragraph 49. A nucleic acid construct comprising the polynucleotide ofparagraph 48.Paragraph 50. An expression vector comprising the nucleic acid constructof paragraph 49.Paragraph 51. A host cell comprising the nucleic acid construct ofparagraph 49.Paragraph 52. A method of producing a variant, comprising:

a. cultivating the host cell of paragraph 51 under conditions suitablefor the expression of the alpha-amylase; and

b. recovering the variant from the cultivation medium.

Paragraph 53. A transgenic plant, plant part or plant cell transformedwith the polynucleotide of paragraph 48.

Paragraph 54. A method for preparing a variant of a parent alpha-amylasecomprising the following steps:

a. providing a nucleic acid encoding a parent alpha-amylase,

b. introducing alterations in the nucleic acid sequence resulting inalterations of the encoded amino acid residues in two, three, four orfive positions, said positions corresponding to positions in the parentalpha-amylase selected from the group consisting of 163, 188, 205, 208and 209; the alteration(s) are independently

-   -   (i) an insertion of an amino acid immediately downstream of the        position,    -   (ii) a deletion of the amino acid which occupies the position,        and/or    -   (iii) a substitution of the amino acid which occupies the        position,

c. optionally introducing further alterations in the nucleic acidsequence resulting in alterations of one or more amino acid residues inthe B-domain of the parent alpha-amylase to the corresponding amino acidresidue(s) in SEQ ID NO: 9;

d. optionally introducing further alterations in the nucleic acidsequence resulting in alterations of the encoded amino acid residuesselected from the group of X183*+X184*, X186A,Y,T, X193F, X195F,M202L,I,T,S,A, X206F,Y, X214I, X244A,D,E,N,Q,W, X452H,Y, X474R andX475R;

e. expressing the altered nucleic acid sequence in a suitable hostorganism; and

f. recovering the variant alpha-amylase,

wherein each position corresponds to a position of the amino acidsequence of the enzyme having the amino acid sequence of SEQ ID NO: 2.

Paragraph 55. The method of paragraph 54, wherein the alterations in b.are selected among: X163Q,N, X188N, X205N, X208Y and, X209N,S.

Paragraph 56. The method of paragraph 54 or 55, wherein the parentalpha-amylase is a Termamyl-like ampha-amylase.

Paragraph 57. The method of paragraph 56, wherein the parentalpha-amylase is selected among alpha-amylases having SEQ ID NO: 2, 4,6, 8 or 10, or alpha-amylases derived from a strain of the Bacillus sp.NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, and the #707alpha-amylase described by Tsukamoto et al., 1988, Biochemical andBiophysical Research Communications 151: 25-31, or alpha-amylases havingat least 70%, preferably at least 75%, more preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least about 97%sequence identity to the amino acid sequence of one of these.

The invention claimed is:
 1. An isolated variant alpha-amylase,comprising two or more alterations at positions corresponding topositions 163, 188, 205, 208, and 209 of the mature polypeptide of SEQID NO: 2, wherein (a) the variant has at least 90% and less than 100%sequence identity to SEQ ID NO: 10; (b) each alteration is independentlya substitution, deletion or insertion; and (c) the variant hasalpha-amylase activity.
 2. The variant of claim 1, which has at least95% sequence identity to SEQ ID NO:
 10. 3. The variant of claim 1,wherein the alterations at positions 163, 188, 205, 208, and 209 aresubstitutions.
 4. The variant of claim 1, wherein the alterations areselected among: X163Q,N, X188N, X205N, X208Y and X209N,S.
 5. The variantof claim 1, further comprising one or more alterations selected from thegroup consisting of a deletion at a position corresponding to positions183 and 184 and a substitution at a position corresponding to thepositions selected from the group consisting of 186, 193, 195, 202, 206,214, 244, 452, 474 and
 475. 6. The variant of claim 1, which furthercomprises one or more alterations selected from the group ofX181*+X182*, X182*+X183*, X183*+X184*, X185K, X167W, X202L/I/T, X203Y,X167W+X168E+X169E+X170R, X51T+X109G+X203Y, X109G+X203Y, X189W,X189W+x190E+x193T, X190E, X193T, X303K,X303K+x305R+x306D+X409N+X432N+X434D, X305R, X306D, X409N, X432N, X434D.7. The variant of claim 1, which further comprises one or morealterations selected from the group consisting of A113E, N116V,V117F,L118K, A119V,V120I,N123D, N126D, N128T, Q129K, G133E, D134P,Y135F, T136E, A139G, D144T, N150D, T151Q, D154S, R158N, W159S, Y160E,V165T, W167F, Q169A, S170K, R171G, Q172*, F173E, Q174R, N175T, R176G,I177V, Y178F, K179R, F180I, R181A, D183E, G184N, A186K, W189E, E190N,S193T/D, N195F, Y203F,V206I, E212D, V214R, and N215R.
 8. The variant ofclaim 1 selected from the group consisting of: D163N+D188N+D209S;D163N+D188N+D205N+D209S; D163N+D188N+D205N+M208F+D209S;D163N+D188N+D207N+D209S; D163N+D188N+D199N+D207N+D209S;D163N+D188N+D199N+D205N+D207N+D209S;D163N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+G186N+D188N+D205N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S;D163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D199N+D205N+M208F+D207N+D209S;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+N409D+D432N+A434P;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W+H408W+N409D+D432N+A434P;N128W+D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+K242P+S244W;D163N+R181A+G182N+K185T+G186N+D188N+D205N+M208F+D209S+V238I+K242P+S244W;and D163Q+D188N+M208F+D209S+K242P+S244W.
 9. The variant of claim 1selected from the group consisting of: D163N+D188N+D209SD163N+D188N+D205N+D209S D163N+D188N+D205N+M208F+D209S D163N+D207N+D209SD163N+D188N+D207N+D209S D163N+D188N+D199N+D207N+D209SD163N+D188N+D199N+D205N+D207N+D209SD163N+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+G186N+D188N+D205N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D205N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D205N+M208F+D209SD163N+R181A+G182N+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+K185T+D188N+D199N+D205N+M208F+D207N+D209SD163N+R181A+G182N+K185T+A186N+D188N+D199N+D205N+M208F+D207N+D209S.
 10. Adetergent composition comprising a variant of claim 1 and a surfactant.